Patients with glioblastoma (GBM) have dismal prognosis and GBM represents an unmet need in medicine. In our search for molecular targets in GBM, we discovered a plasma membrane receptor, interleukin 13 receptor alpha 2 (IL-13RA2), which is over-expressed in vast majority of patients (protein: >70%; gene: 58%), but not in normal brain. The receptor is over-expressed preferentially in a mesenchymal subgroup of GBM and may be also important for glioma stem-like cells pathobiology. Thus far, this receptor has served clinical development of several therapeutic approaches, because it is a very specific and attractive target for diagnosing, imaging and treatment of IL-13RA2 expressing malignancies, including melanoma and other aggressive peripheral tumors. With the help of this currently held grant, we have generated novel agents that recognize IL-13RA2 and they are amenable to translational efforts in order to better manage cancer. Firstly, in proof-of-concept study, a double-level targeted agent, which is composed of IL-13RA2 receptor-recognition unit (IL-13.E13K, a mutated form of native ligand), a portion of a bacterial toxin that enables cytosolic transport (D2) and a nuclear localization signal (NLS) IL-13.E13K-D2-NLS-Cys, was conjugated to a modified Doxorubicin (WP936). IL- 13.E13K-D2-NLS-Cys[WP936] conjugate showed distinct advantages over the treatment with WP936 alone on GBM cells. This opens a novel way of delivering drugs/labels not only to a targeted subpopulation of cells, but also directly to their intracellular site of action. Secondly, we have isolated a small peptide, Pep-1-L which binds specifically IL-13RA2, induces its internalization and the binding is not competed for by either native ligand or anti-IL-13RA2 antibody. We have also demonstrated that the peptide home to peripheral and intracranial tumors in mice when injected systemically. Thirdly, we have raised monoclonal antibody 1E10B9, which recognizes specifically IL-13RA2 in various immunoreactivity assays in tissue specimens and live cells. We plan on taking a full advantage of these novel agents for the purpose of monitored effective treatment of malignancies over-expressing IL-13RA2 by executing three Specific Aims. In Aim 1, we will continue developing drug conjugates with intrinsic property of delivering drugs to their site of action in cancer cells We will use a blood-brain barrier-penetrating anthracyclin, Berubicin, for conjugation to our novel drug delivery vectors. In Aim 2, we will exploit small peptides binding to IL-13RA2 for double-load combinatorial targeted therapy and monitoring of responses to therapy. In Aim 3, we will generate recombinant fragments of antibody 1E10B9, optimize its binding, and equip with ADCC/CDC and CTLA4 or PD1 immune checkpoints blocking activity. We will also study the structural basis of Pep-1-L and 1E10B9 binding to IL-13RA2. The pre-clinical evaluation of these candidate drugs/imaging agent will be conducted in models of GBM and melanoma. We expect to generate novel, specific and potent therapeutics with a possibility to monitor their effectiveness against some of the most dreadful human malignancies, to be examined in patients in a foreseeable future.